Ethanol pulping is a kind of solvent pulping technology. The utilization of lignin in waste liquor can reduce pollution and increase high value of product. The lignin in the waste liquor could be isolated by adding water in the system after standing for several hours and purified by enzymatic and acidic hydrolysis two-step procedure. Results showed that the optimized purification conditions are as follows, enzyme dosage is 0.004g/g (to oven dry lignin), enzymatic buffer pH is 5, enzymatic hydrolysis temperature is 30°C, and hydrochloric acid concentration of acid hydrolysis is 0.055mol/L. The lignin purity is 91.61%, and the yield is 81.89%.